The quantitative determination of analytes in biological fluids such as whole blood is of great importance in the diagnosing and treating of certain medical conditions. For example, determining the glucose level in the blood of diabetic individuals, who must frequently check the glucose level in their blood to regulate their diets and medication. Measuring the glucose content of blood can be done by several methods. One method employs an electrochemical biosensor that relates glucose content to a measured electrical current. Another method provides a visual indication of the glucose content, such as by developing a color by reaction of an indicator. While the present invention is particularly useful in optical measurements, it also has application to electrochemical biosensors.
There have been many patents describing methods employing indicators that develop color or other measurable responses when they are chemically oxidized as the last step of a series of reactions. For example, methods that employ enzymes, such as, analyte oxidases (e.g. glucose oxidase) or analyte dehydrogenases (e.g. glucose dehydrogenase). The procedures used are similar, but they employ different enzymes, mediators and indicators.
Methods using glucose oxidase enzymes are taught in many US patents and patent applications. Representative are U.S. Pat. Nos. 4,211,845; 4,808,529; 5,116729; 5,264,348; 5,620,863; and 2003/0077702 A1. These patents teach a method in which glucose is oxidized to gluconic acid with the release of hydrogen peroxide. The hydrogen peroxide is said to oxidize an indicator in the presence of a peroxidase to produce a measurable color, indicating the glucose content of the blood sample. Some recent patents suggest a process in which the glucose is converted first to gluconic acid and then to gluconolactone with the release of hydrogen peroxide. It has also been suggested that the gluconolactone is formed first and then hydrolyzed to gluconic acid. Regardless of which process scheme is correct, glucose oxidase enzymes have been used widely in dry strips and in other techniques for measuring the glucose content of blood.
Various indicators have been employed in glucose sensors, such as benzidine-type indicators and heterocyclic azines. For example, 3,3′,5,5′-tetramethylbenzidine and syringaldazine, luminol, o-tolidine, o-dianisitine, among others. Another family of indicators is tetrazolium dye precursors. Examples of patents describing such indicators include U.S. Pat. Nos. 5,126,275, 5,322,680, 5,300,637, 5,290,536, 5,360,595 and 6,586,199. Tetrazolium indicators are used in a preferred embodiment of the invention to be described below.
Of particular interest with regard to the present invention is the method described in U.S. Pat. No. 6,200,773 and its parent U.S. Pat. No. 5,902,731. In these patents, a test of the glucose content of blood employs glucose dehydrogenase, as a cofactor NAD or PQQ or their derivatives, a tetrazolium dye precursor, a diaphorase enzyme or an analog, and a nitrite salt. FIG. 5 of the '773 patent is a diagram of the process by which glucose is detected by development of color from the reduction of the tetrazolium dye precursor to formazan.
An early patent relating to the use of enzymes to determine the amount of glucose in blood is U.S. Pat. No. 3,630,957. Glucose oxidise and peroxidase were uniformly distributed in a water-resistant polymer film to react with glucose and to produce a color. The film could be supported on a substrate, e.g. a polymer film. It was suggested that fillers including chalk, titanium dioxide, colloidal silicic acid (used in the examples) and the like could be added and that pigments could be included to make the films opaque. Blood was applied to the reagent-containing film and then wiped off before reading the developed color. The use of opaque fillers to reduce interference with glucose measurements by red blood cells was also discussed in U.S. Pat. No. 5,968,765.
Another patent of interest is U.S. Pat. No. 4,312,834, which describes the use of a water-resistant film including fine insoluble particles, which provide access to the reagents while blocking access of large components. The film could be supported on carriers, such as films, foils, etc. The patentees were concerned with access of certain molecules, stating that the amount of the fine particles (termed “film openers”) should be with certain limits. Various types of particles were suggested, such as kieselguhr gel, silica gel, and gypsum, and the like. Titanium dioxide was suggested, both as a film opener and as a way to improve the remission properties of the film. In general, relatively thick films of 200-400 μm were deposited on substrate films in the examples; in some cases multiple layers were applied. As in the '957 patent, excess of the sample, e.g. blood, was wiped off after the reaction had occurred.
Test strips have been described in many patents, since they are widely used for detection of analytes in biological samples. Each test strip application has its unique problems which must be overcome if accurate and consistent results are to be obtained. Testing whole blood requires that the red blood cells not interfere with the color which is developed to indicate the presence of glucose, or with electrochemical measurements. In some instances specific components are included in the test strips so that the red blood cells are filtered from the sample. In other cases the sample is wiped off after a period of time has elapsed so that the developed color can be measured. Another problem encountered when testing whole blood relates to the concentration of red blood cells in the sample. They are commonly measured by their volume in the sample and referred to as the hematocrit value. Since the hematocrit may vary from 20 to 60% in blood samples, the glucose measurement may be affected. Also, movement of the blood plasma carrying glucose to the reagents to develop color (or an electrochemical response), may be retarded or incomplete.
Preventing red blood cells from reaching the reagents that react with glucose has been a concern of many workers in the art. In the test strip of the '765 patent mentioned above, to a 0.002 to 0.2 inch (50.8 to 5080 μm) thick porous membrane was added an agent for separating red blood cells from whole blood, which included polyacrylic acid among others, an indicating reagent and an opaque filler, e.g. titanium dioxide, talc, etc. The coating solution was deposited on the surface of the porous membrane or imbibed within the membrane.
In U.S. Pat. No. 5,306,623 a coating capable of separating whole blood was selected from a group of polymers including polyvinyl sulfonic acid, polyethylene glycol, polystyrene sulfonic acid, hydroxypropyl cellulose, polypropylene glycol, polyvinyl pyrrolidone and polyacrylic acid. The separating coating was deposited on a porous matrix along with reagents for testing blood.
Sensitivity of blood test strips to the hematocrit of whole blood was discussed in U.S. Pat. No. 5,789,255, The inventor found that addition 0.1-2% w/v of a high molecular weight (>750,000) acrylic acid polymer reduced the effect of varying hematocrit on the glucose measurements.
The ideal test strip for measuring glucose in whole blood samples would be insensitive to the hematocrit of the blood sample and provide rapid, accurate, and consistent results. A fast response time combined with a stable endpoint would provide a test that is significantly less time dependent and therefore more convenient in the hands of the user. In another aspect of importance to the user, the test strip should be insensitive to the volume of blood applied. The test strip to be described in more detail below closely approaches that ideal performance.